PROJECT SUMMARY Cell surface and secreted glycoproteins form a complex interface with the extracellular environment that influences cellular differentiation, physiology, and pathology. Very little is known about how glycan diversity is controlled to produce distinct sets of glycan structures in different cell types or on individual glycoproteins. Glycan structures are synthesized by the action of glycosyltransferases (GTs) that yield heterogeneous ensembles of glycan structures on each site of a given glycoprotein. Challenges remain in defining the underlying `rules' that specify selective, site-specific modification of glycoproteins, including: 1) deciphering how individual glycoenzyme active sites act as templates to specify regiospecific substrate recognition and 2) determining how the context and steric constraints of individual glycosylation sites (glycosites) can limit or restrict access to tune the diversity of glycan structures produced. We assembled an integrated research team with expertise in glyco-enzymology, recombinant glycoprotein expression, glyco-analytical chemistry, protein structural biology, bioinformatics, and chemo-enzymatic glycan synthesis to leverage our unique toolsets and expertise to identify the essential features that govern site-specific glycan diversity. Our aims include (Aim 1) determining how glycoenzyme active sites provide templates for glycan modification. We will pursue structural studies on enzymes in complex with donor analogs and synthetic glycan acceptors and leverage bioinformatic analyses to generate new hypotheses regarding the evolution of glycoenzyme substrate recognition and specificity. These hypotheses will be tested by mutagenesis, protein redesign, and enzyme activity toward acceptor substrates. In Aim 2 we will determine the structural basis for site-specific modification of glycoprotein acceptors by examining the efficiency of enzymatic modification through the use of MS-based glycopeptide mapping approaches. Structural data for the respective reporter glycoproteins will be used to compare glycosite modification with steric constraints for individual glycosites. Hypotheses regarding glycosite accessibility will be tested by mutagenesis of enzyme active sites and regions that flank the glycosites on the glycoprotein reporters. In Aim 3 we will test our hypotheses for site-specific glycan modifications by reporter expression in cultured cells. Site-specific glycoforms produced on the glycoprotein reporters in the mammalian secretory pathway will be examined to determine if biosynthetic `rules' identified from in vitro studies will extend to glycan modifications in the more complex environment of the cellular secretory pathway. The proposed studies will provide fundamental knowledge on how glycoenzymes act as templates for the creation of diverse glycan structures and how the steric constraints of their substrates tune those specificities to provide predictable glycan diversity on individual glycan sites.